Available online />Page 1 of 7
(page number not for citation purposes)
Abstract
One of the major challenges in rheumatology is to overcome the
classification criteria that previously defined systemic lupus
erythematosis, since the heterogeneity of the disease(s) appears to
represent a complexity that probably substantially contributed to
the failure of a number of recent trials. For those engaged in clinical
trials, validated disease activity biomarkers that respond rapidly to
treatment and are predictive of clinical response would greatly
facilitate early decision-making around futility and dose selection.
Likewise, use of validated patient stratification biomarkers possibly
in conjunction with autoantibody profiles and disease manifes-
tations will result in the recruitment of more homogeneous patient
populations during later stage clinical studies, thereby decreasing
size, costs, and risks in pivotal studies.
Challenge of lupus for drug development
Systemic lupus erythematosis (SLE) is perhaps the most
clinically and serologically diverse of the autoimmune diseases.
The current American College of Rheumatology classification
lists 11 criteria for diagnosis of lupus, of which a patient must
meet four [1]. The heterogeneity of the patient population
results in significant challenges not only in classifying disease
activity but also for establishment of therapeutic response to
new drug candidates and therapeutic strategies.
Outcome measures used in clinical trials currently rely on one
(or more) of several disease activity indices – the Systemic
Lupus Erythematosis Disease Activity Index (SLEDAI), the
Systemic Lupus Activity Measure, the British Isles Lupus
Assessment Group (BILAG), the European Consensus
Lupus Activity Measure – and their derivatives. These tools

immunological signature as part of the lupus classification
criteria could aid in evaluation of novel therapeutics, and
ultimately in treatment decision-making.
Review
Biomarkers as tools for improved diagnostic and therapeutic
monitoring in systemic lupus erythematosis
Michael F Smith, Jr
1,2
, Falk Hiepe
3
, Thomas Dörner
3
and Gerd Burmester
3
1
Wyeth Research, Discovery Translational Medicine, Collegeville, PA 19426, USA
2
Present address: Hoffmann-La Roche, Inc., 340 Kingsland Street, Building 721, Nutley, NJ 07110, USA
3
Universitätsmedizin Charité Berlin, 10098 Berlin, Germany
Corresponding author: Michael F Smith,
Published: 19 November 2009 Arthritis Research & Therapy 2009, 11:255 (doi:10.1186/ar2834)
This article is online at />© 2009 BioMed Central Ltd
BAFF = B-cell activating factor of the TNF family; BILAG = British Isles Lupus Assessment Group; CNS = central nervous system; dsDNA =
double-stranded DNA; ICOS = inducible costimulator; IFN = interferon; IL = interleukin; sCD25 = soluble IL-2 receptor; Siglec-1 = sialic acid
binding immunoglobulin-like lectin 1; SLE = systemic lupus erythematosis; SLEDAI = Systemic Lupus Erythematosis Disease Activity Index; TNF =
tumor necrosis factor.
Arthritis Research & Therapy Vol 11 No 6 Smith et al.
Page 2 of 7
(page number not for citation purposes)

measurements of renal disease. These designs typically call
for 6-month to 12-month clinical endpoint analyses of renal
response (if not even longer, as could be concluded from the
recent Rituximab clinical trial in lupus nephritis (LUNAR)).
Because of increased competition in the lupus field, this
patient population will be increasingly difficult to recruit – as a
result, the expected length of the proof-of-concept study may
be upwards of 2 years or more. Validated disease activity
biomarkers that respond rapidly to treatment and are
predictive of clinical response at later time points could greatly
facilitate early decision-making around futility and dose
selection, thereby shortening potentially lengthy proof-of-
concept studies. Furthermore, such biomarkers would
enhance the development of adaptive trial designs, something
not currently possible in lupus nephritis trials, further
streamlining the clinical trial process.
An additional advantage to developing a well-validated
biomarker toolbox will be the potential to identify patients
during early phase studies who are likely to respond to the
treatment being tested and who have a higher likelihood to
achieve a major response or even remission. Such patient
stratification biomarkers can result in the recruitment of a
more homogeneous patient population during later stage
clinical studies with the promise of decreasing size, costs,
and risks in pivotal registration studies.
New approaches to lupus disease activity
biomarkers
Complement levels and anti-dsDNA antibodies are classic
biomarkers of lupus disease activity that have been shown in
some, but not all, studies to be predictors of outcome in

with anti-RO (SS-A) production related to the HLA-DQ1/
DQ2 heterozygotes, anti-La (SS-B) related to HLA-B8 and
HLA-DR3, and anti-nuclear RNP (Sm) related to HLA-DR4.
While lymphopenia was associated significantly with anti-Ro
(SS-A) and, secondarily, with anti-single-stranded DNA, lupus
nephritis was inversely associated with anti-La (SS-B) and
associated with anti-dsDNA. It has been repeatedly shown
that anti-dsDNA, single anti-Ro antibodies as well as Sm
antibodies [10] are associated with lupus nephritis, and in
part with central nervous system (CNS) lupus, whereas com-
bined anti-Ro/La antibodies are associated with secondary
Sjögren’s syndrome and photosensitivity, absence of lupus
nephritis and severe CNS involvement. Moreover, anti-
ribosomal P antibodies are clearly associated with CNS lupus
[11], and anticardiolipin antibodies mark patients with throm-
botic events as well as thrombocytopenia [12], serving as
reliable identifiers of these clinical presentations. In a very
instructive study, the occurrence of anti-U1RNP/Sm
antibodies occurred early on and prior to the disease onset
as well as at different times from other autoantibodies [13].
These data indicate that genetic background, including HLA
class II, is important for the induction of certain auto-
antibodies that contribute to the clinical heterogeneity and
variation in disease outcomes among SLE patients. The
established associations of autoantibody profiles with clinical
subtypes may at least require consideration in the design of
SLE trials for defining stricter outcome criteria.
The interferon signature
One of the most promising lupus disease activity biomarkers
to be identified in recent years is the so-called IFN signature.

in the patient compared with a healthy donor group. Such a
composite score can therefore decrease variability and
potentially be more indicative of real changes in disease
activity. This approach, however, requires each laboratory to
develop its own unique panel of genes and validate the assay
with a population of healthy controls and lupus patients.
Standardization of an IFN gene signature profile amongst
investigators would help significantly for comparing results
between studies.
The correlation of the IFN signature with more traditional
measures of lupus nephritis activity was evaluated by Bauer
and colleagues [25]. Thirty patients were classified as having
high or low IFN gene scores (15 patients each) and the
association with lupus disease activity indices or other clinical
features was determined. High IFN scores were positively
associated with increased disease activity as indicated by the
SLEDAI and the Systemic Lupus Activity Measure – Revised
but not by the BILAG or physician global assessment.
Immunologic manifestations and decreased C3 were also
associated with high IFN signatures. Similarly, Kirou and
colleagues, also found significant correlations between IFN
scores, the SLEDAI 2000 immunologic manifestations, and
decreased C3 [26]. In addition, this group also identified
significant associations between renal involvement and
autoantibody profiles.
Similar associations between disease activity scores and IFN
scores have been found in all studies that examined them.
Importantly, one study also demonstrated that treatment of
lupus patients with high-dose steroids rapidly extinguished
the IFN gene signature, suggesting that this biomarker may

described above with lupus nephritis, it is perhaps not
surprising that a protein correlate of this activity can be found
Available online />Page 3 of 7
(page number not for citation purposes)
in the serum of this patient subset. Bauer and colleagues
used a multiplex serum protein analysis to measure the levels
of 160 different proteins in the serum of lupus patients and of
healthy control subjects [25]. From this original panel, 30
analytes were identified that were dysregulated in lupus
nephritis patients – 12 of which were determined by in vitro
studies to be IFN regulated. Notably, CD25 was also part of
this serum signature. As with the IFN gene signature, the
composite chemokine protein score positively correlated with
the SLEDAI, with the Systemic Lupus Activity Measure –
Revised, and with anti-dsDNA antibodies. Lupus patients in
this study were also grouped according to the IFN gene
signature score (high vs. low), and the cytokine/chemokine
profiles between the IFN high and IFN low groups were
demonstrated to be remarkably similar and both were very
distinct from healthy controls. When correlations between
individual chemokines and specific organ manifestations were
examined, there appeared to be differences in the serum
chemokine profiles between the different groups. In
particular, it is interesting to note that there was actually a
negative correlation of chemokine levels to the presence of
hematological manifestations (predominantly thrombocyto-
penia). Although sample sizes in this study were very small
(n = 7 for hematologic samples), this finding is nevertheless
very intriguing – and if confirmed in larger studies, may
provide additional means for stratifying patients to potential

serum of SLE patients have been described for 20 years
[30-32]. In addition, soluble forms of CD27 and CD40 ligand
have also been detected in serum from lupus patients
[33,34]. While most studies have demonstrated a positive
correlation between levels of sCD25 and disease activity,
longitudinal studies have also supported its use as a
biomarker that is closely correlated with flare activity and
therapeutic responses, particularly in patients with renal
involvement [35,36]. sCD25 does not, however, appear to be
a highly responsive marker in patients with moderate disease
or mild flare.
In total, the results of studies into the association of sCD25
with lupus disease activity strongly support the inclusion of
this protein as part of a biomarker panel. As with the
measurement of complement activation, however, its utility
may be limited to studies of patients with high disease
activity.
B-cell activating factor of the TNF family
B-cell activating factor of the TNF family (BAFF) is a cytokine
belonging to the TNF superfamily, and is a B-cell activator
that controls peripheral B-cell maturation. BAFF (also known
as BLys) stimulates B-cell proliferation and is necessary for
B-cell survival. Transgenic overexpression of BAFF in mice
results in abnormally high B-cell numbers and in the develop-
ment of a lupus-like disease [37]. This observation suggested
a role for BAFF in SLE and was further evaluated in patients.
Serum levels of BAFF and BAFF mRNA in peripheral
leukocytes are found to be elevated in lupus patients and are
positively associated with disease activity indices [38-41]. Of
note, BAFF levels tend to fluctuate during the natural course

and response to treatment.
Inducible costimulator expression
The inducible costimulator (ICOS) is a member of the CD28
family that, like CD28, can enhance T-cell proliferation and
cytokine secretion. In the first study in human SLE, Hutloff
and colleagues demonstrated overexpression of ICOS on
CD4
+
and CD8
+
T cells and a remarkable reduction of ICOS
ligand on CD27
+
memory B cells [47]. In addition, lupus
nephritis patients had an accumulation of germinal center-like
structures in their kidneys. These data suggested that there is
continuous crosstalk and activation between T cells and B
cells in SLE, leading to the overactivation of the adaptive
immunity in lupus.
In confirmation, Yang and colleagues investigated the
expression of ICOS on CD4 and CD8 T cells from lupus
patients [48] in peripheral blood from moderately and highly
active SLE patients. They found elevated numbers of ICOS-
positive T cells, both CD4 and CD8, as well as elevated
levels of ICOS expression compared with healthy controls or
rheumatoid arthritis patients. Furthermore, longitudinal analy-
sis of individual patients indicated that ICOS was significantly
elevated during the active phase when compared with clinical
remission.
Sialic acid binding immunoglobulin-like lectin 1

of lupus. Jacobi and colleagues evaluated the correlation of
CD27
high
plasma cells and disease activity in lupus patients
[51]. Patients with high disease activity (SLEDAI >8) had
significantly increased frequency of CD19
+
/CD27
high
plasma
cells and had a predictive value for disease activity that was
greater than traditional humoral/clinical measurements (for
example, anti-dsDNA, complement, renal manifestations). In
addition, there was a statistically significant correlation
between these plasma cells with the SLEDAI as well as with
the anti-DNA titers. Interestingly, the CD27
high
cells also
correlated with the SLEDAI as measurement of the disease
activity in SLE in patients not producing anti-dsDNA
autoantibodies. More recent data show that a particular
CD27
–
/IgD
–
B-cell subset is expanded in SLE [52,53]. Only
the CD27
–
/IgD
–

within clinical trials. In order for a biomarker to be qualified for
use in decision-making, epidemiologic or observational
studies of the natural history of the disease should establish
the relationship between the biomarker, defined clinical
cohorts as already widely used for lupus nephritis, and
defined clinical endpoints.
To date, the vast majority of biomarker studies in lupus have
attempted to link clinical activity to only one, or a few,
Available online />Page 5 of 7
(page number not for citation purposes)
potential biomarkers. Given the heterogeneity of the disease –
is lupus really one disease? – a multiplexed approach is
clearly going to be essential. Biomarker validation is an
iterative process requiring multiple studies to identify,
characterize, and confirm the validity of the biomarker for the
intended purpose. Pharmaceutical companies and clinical
trialists need to commit to the evaluation of multiple,
exploratory biomarkers within clinical studies and to sharing
of the information if we are to improve our understanding of
lupus and the response to active therapies. It is a long-term
investment but one that the lupus community has recognized
and is embracing as the critical path to a successful lupus
therapy.
Competing interests
The authors declare that they have no competing interests.
References
1. Tan EM, Cohen AS, Fries JF, Masi AT, McShane DJ, Rothfield NF,
Schaller JG, Talal N, Winchester RJ: The 1982 revised criteria
for the classification of systemic lupus erythematosus. Arthri-
tis Rheum 1982, 25:1271-1277.

T, Schneider-Mergener J, Burmester GR, Hiepe F: A novel
epitope on the C-terminus of SmD1 is recognized by the
majority of sera from patients with systemic lupus erythe-
matosus. J Clin Invest 1998, 102:754-763.
11. Shoenfeld N, Agmon-Levin N, Flitman-Katzevman I, Paran D, Katz
BS, Kivity S, Langevitz P, Zandman-Goddard G, Shoenfeld Y: The
sense of smell in systemic lupus erythematosus. Arthritis
Rheum 2009, 60:1484-1487.
12. Krause I, Blank M, Fraser A, Lorber M, Stojanovich L, Rovensky J,
Shoenfeld Y: The association of thrombocytopenia with sys-
temic manifestations in the antiphospholipid syndrome.
Immunobiology 2005, 210:749-754.
13. Arbuckle MR, McClain MT, Rubertone MV, Scofield RH, Dennis
GJ, James JA, Harley JB: Development of autoantibodies
before the clinical onset of systemic lupus erythematosus.
N Engl J Med 2003, 349:1526-1533.
14. Hooks JJ, Moutsopoulos HM, Geis SA, Stahl NI, Decker JL,
Notkins AL: Immune interferon in the circulation of patients
with autoimmune disease. N Engl J Med 1979,
301:5-8.
15. Vallin H, Blomberg S, Alm GV, Cederblad B, Rönnblom L:
Patients with systemic lupus erythematosus (SLE) have a cir-
culating inducer of interferon-alpha (IFN-
αα
) production acting
on leucocytes resembling immature dendritic cells. Clin Exp
Immunol 1999, 115:196-202.
16. Vallin H, Perers A, Alm GV, Rönnblom L: Anti-double-stranded
DNA antibodies and immunostimulatory plasmid DNA in com-
bination mimic the endogenous IFN-alpha inducer in systemic

Johnson D, Lanier A, Punaro M, Wittkowski KM, White P, Fay J,
Klintmalm G, Ramilo O, Palucka AK, Banchereau J, Pascual V: A
modular analysis framework for blood genomics studies:
application to systemic lupus erythematosus. Immunity 2008,
29:150-164.
25. Bauer JW, Baechler EC, Petri M, Batliwalla FM, Crawford D,
Ortmann WA, Espe KJ, Li W, Patel DD, Gregersen PK, Behrens
TW: Elevated serum levels of interferon-regulated
chemokines are biomarkers for active human systemic lupus
erythematosus. PLoS Med 2006, 3:e491.
26. Kirou KA, Lee C, George S, Louca K, Peterson MG, Crow MK:
Activation of the interferon-alpha pathway identifies a sub-
group of systemic lupus erythematosus patients with distinct
serologic features and active disease. Arthritis Rheum 2005,
52:1491-1503.
27. Biesen R, Demir C, Barkhudarova F, Grun JR, Steinbrich-Zollner
M, Backhaus M, Haupl T, Rudwaleit M, Riemekasten G, Radbruch
A, Hiepe F, Burmester GR, Grutzkau A: Sialic acid-binding Ig-
like lectin 1 expression in inflammatory and resident mono-
cytes is a potential biomarker for monitoring disease activity
and success of therapy in systemic lupus erythematosus.
Arthritis Rheum 2008, 58:1136-1145.
28. Yao Y, Richman L, Higgs BW, Morehouse CA, de Los Reyes M,
Brohawn P, Zhang J, White B, Coyle AJ, Kiener PA, Jallal B: Neu-
tralization of interferon-alpha/beta-inducible genes and
downstream effect in a phase I trial of an anti-interferon-alpha
monoclonal antibody in systemic lupus erythematosus. Arthri-
tis Rheum 2009, 60:1785-1796.
29. Bauer JW, Petri M, Batliwalla F, Wilson JC, Koeuth T, Singh S,
Tesfasyone H, Gregersen PK, Behrens TW, Baechler EC: Base-

αα
, p75srTNF
αα
, srIL-2
αα
levels and disease activity in systemic lupus erythematosus.
Clin Rheumatol 1999, 18:17-22.
36. ter Borg EJ, Horst G, Limburg PC, Kallenberg CG: Changes in
plasma levels of interleukin-2 receptor in relation to disease
exacerbations and levels of anti-dsDNA and complement in
systemic lupus erythematosus. Clin Exp Immunol 1990, 82:21-
26.
37. Khare SD, Sarosi I, Xia XZ, McCabe S, Miner K, Solovyev I,
Hawkins N, Kelley M, Chang D, Van G, Ross L, Delaney J, Wang
L, Lacey D, Boyle WJ, Hsu H: Severe B cell hyperplasia and
autoimmune disease in TALL-1 transgenic mice. Proc Natl
Acad Sci U S A 2000, 97:3370-3375.
38. Becker-Merok A, Nikolaisen C, Nossent HC: B-lymphocyte acti-
vating factor in systemic lupus erythematosus and rheuma-
toid arthritis in relation to autoantibody levels, disease
measures and time. Lupus 2006, 15:570-576.
39. Ju S, Zhang D, Wang Y, Ni H, Kong X, Zhong R: Correlation of
the expression levels of BLyS and its receptors mRNA in
patients with systemic lupus erythematosus. Clin Biochem
2006, 39:1131-1137.
40. Lin Z, Dan-Rong Y, Xiao-Qing T, Hao W, Hong-Lian G, Xian-Tao
K, Ren-Qian Z, Jiyu L: Preliminary clinical measurement of the
expression of B-cell activating factor in Chinese systemic
lupus erythematosus patients. J Clin Lab Anal 2007, 21:183-
187.

systemic lupus erythematosus. Arthritis Rheum 2004, 50:
3211-3220.
48. Yang JH, Zhang J, Cai Q, Zhao DB, Wang J, Guo PE, Liu L, Han
XH, Shen Q: Expression and function of inducible costimula-
tor on peripheral blood T cells in patients with systemic lupus
erythematosus. Rheumatology (Oxford) 2005, 44:1245-1254.
49. Hartnell A, Steel J, Turley H, Jones M, Jackson DG, Crocker PR:
Characterization of human sialoadhesin, a sialic acid binding
receptor expressed by resident and inflammatory
macrophage populations. Blood 2001, 97:288-296.
50. van den Berg TK, Nath D, Ziltener HJ, Vestweber D, Fukuda M,
van Die I, Crocker PR: Cutting edge: CD43 functions as a T cell
counterreceptor for the macrophage adhesion receptor
sialoadhesin (Siglec-1). J Immunol 2001, 166:3637-3640.
51. Jacobi AM, Odendahl M, Reiter K, Bruns A, Burmester GR, Rad-
bruch A, Valet G, Lipsky PE, Dorner T: Correlation between cir-
culating CD27
high
plasma cells and disease activity in patients
with systemic lupus erythematosus. Arthritis Rheum 2003, 48:
1332-1342.
52. Jacobi AM, Reiter K, Mackay M, Aranow C, Hiepe F, Radbruch A,
Hansen A, Burmester GR, Diamond B, Lipsky PE, Dorner T: Acti-
vated memory B cell subsets correlate with disease activity in
systemic lupus erythematosus: delineation by expression of
CD27, IgD, and CD95. Arthritis Rheum 2008, 58:1762-1773.
53. Wei C, Anolik J, Cappione A, Zheng B, Pugh-Bernard A, Brooks J,
Lee EH, Milner EC, Sanz I: A new population of cells lacking
expression of CD27 represents a notable component of the B
cell memory compartment in systemic lupus erythematosus.